Improved GAL4 and Tet OFF drivers for C. elegans bipartite expression

The first generation of C. elegans GAL4 drivers for bipartite expression function less well than C. elegans tet ON/OFF, QF and LexA drivers. The main difference between the GAL4 drivers and the others is the absence of a flexible linker between the DNA binding and activation domain in the GAL4 construct. Addition of a linker to a GAL4-QF construct increased driver potency, while adding linkers to a GAL4-VP64 driver was much less effective. Extending the linker region of the tetR-L-QF driver also increased activity of that driver. The new GAL4 driver makes GAL4/UAS bipartite system activity comparable to the other worm bipartite expression systems.

. GAL4 SK and TetR bipartite driver activity improves with a flexible linker: Quantification of GFP expression of various GAL4 SK and TetR bipartite drivers with distinct linkers separating the DNA and activation domains. The mec-4 promoter was used to express the drivers. An 11X UAS ∆pes-10p GFP-C1 reporter was used to assay GAL4 SK activity and a 7X tetO ∆pes-10 GFP-C1 reporter was used to assay TetR activity. See strain list for the exact genotype of animals analyzed. Individual measurements (filled grey circles) and the mean (black bar) are shown. The units are defined identically for ALM and PLM measurements. All of the transgenes also express in AVM and PVM. None of the transgenes express in any other cell types to detectable levels. Strains used: NM5225, NM5467, NM5468, NM5301, NM5470, NM5233 and NM5362. n=15-17 for ALM and 30-34 for PLM. T-test: p*<0.01, ** p<0.0001.

Description
Several bipartite systems have been described for use with C. elegans (Wei et al., 2012;Wang et al., 2017;Wang et al., 2018;Mao et al., 2019;Nonet, 2020). However, little manipulation of the drivers or reporters has been performed to optimize them for C. elegans. Mao. et. al. 2019 demonstrated that the QF activation domain is a more potent activator than either VP64 or the hybrid VP64-p65-Rta tripartite activatorVPR. Here, I describe modifications of linker region between the activation domains of both GAL4 SK and TetR drivers that increase the activity of these reporters. Although these studies are not comprehensive, I opted to describe them herein because the modification of the GAL4 SK driver increases the activity of this driver substantially such that it is now on par with the LexA, TetR and QF2 drivers.
I used an efficient RMCE protocol (Nonet, 2020) to create the transgenic animals. Modified versions of a mec-4 promoter GAL4 SK -QF AD , GAL4 SK -VP64 and TetR-QF AD constructs were created in an RMCE integration vector using a Golden Gate cloning approach, then integrated on Chr IV using a standard injection protocol. After outcrossing to the appropriate reporter, the expression level of GFP at steady state in PLM and ALM soma of L4 animals was quantified ( Figure 1).
Previously, I described drivers consisting of C. elegans codon optimized synthetic GAL4 SK , TetR and LexA DNA binding domains fused to the QF activation domain based on the observations of Mao. et al. 2019. Although the GAL4 SK construct was modestly active, the LexA and TetR construct were incapable of activating the reporter (supplemental methods of Nonet, 2020). Replacement of a 49 amino acid portion of central domain of QF, which separates the TetR and LexA DNA binding domains and the QF activation domain in the original constructs, with a 12 amino acid flexible linker converted both into much stronger drivers than the GAL4 SK -QF AD driver (Nonet, 2020). Here I show that insertion of a similar linker also greatly increases the potency of the GAL4 SK -QF AD driver. I also extended the linker of the TetR-L-QF AD and this further improved activity of this driver. In Nonet, 2020, I also tested the functionality of a GAL4 SK -VP64 construct (Wang et al. 2017) in single copy and found it was incapable of expressing the GFP reporter. To test if the failure of GAL4 SK -VP64 was also the result of insufficient domain separation, I inserted a 40 amino acid flexible linker in between GAL4 and VP64. Although the linker containing driver activates transcription, it does so extremely poorly in comparison to the GAL4 SK -QF AD drivers. I speculate this is likely due to the loss of the MED25 subunit of the mediator complex in the nematode lineage (Grants et al., 2015). VP64 (a 4X VP16) is known to activate transcription in part through interaction with MED25 (Mittler et al., 2003) and the loss of this interaction could account for the observed weak activation properties of VP64 and related activators in worms (Mao et al. 2019 and herein).
In addition to the modification of the linker domain of the transgenes I characterize herein, some of the transgenes also differ in other ways that could theoretically impact my conclusions. First, some driver transgenes contain a tbb-2 3′ UTR and others use an act-4 3′ UTR. I consider it very unlikely these differences impact GFP expression for two reasons. First, my lab has previously demonstrated that mec-4promoter driven GFP-C1 transgenes employing the tbb-2 3′ UTR and the act-4 3′ UTR express at very similar levels in ALM and PLM (Dour and Nonet, 2021). More importantly, I previously demonstrated that the activity of the both a GAL4-QF AD driver and a LexA-L-QF AD driver are insensitive to dosage of the driver ( Figure 5 of Nonet, 2020). Specifically, GFP levels observed in GAL4 SK /+; UAS::GFP ~= GAL SK ; UAS::GFP. Thus, the level of expression of the driver is unlikely to be determining the GFP signal level. Rather, I speculate that GAL4 SK is saturating the UAS binding sites in all transgenes and that the inherent activation properties of the driver determines the expression level.
The improvements to GAL4 SK -QF AD by insertion of a flexible linker is an important addition to the C. elegans bipartite expression toolkit since the GAL4 system is so extensively developed in Drosophila. Using multi-copy lines and a VP64 activator, Wang et al. (2018) have already shown that the split GAL4 SK system is functional in worms. Incorporation of a similar flexible linker and a QF activation domain into those tools should permit development of a robust single copy split-GAL4 SK system. Other GAL4 system tools previously developed for Drosophila such as GAL80ts and GAL4-PR tools (Caygill and Brand, 2016) which provide temporal control in addition to spatial control could also easily be incorporated into the worm toolbox.
In addition, further manipulation of the size and properties of the linker domain separating the DNA binding domain and activation domains could yield drivers with even stronger activation proper which would likely also be applicable to the LexA, QF and Tet ON/OFF driver/reporter systems.

Methods
C. elegans was maintained on NGM agar plates spotted with OP50 at 22.5°C or at 25°C during the RMCE protocol.

Microscopy
For quantification of GFP signals, homozygous L4 hermaphrodite animals were mounted on 2% agar pads in a 2 µl drop of 1mM levamisole in phosphate buffered saline and imaged on an Olympus (Center Valley, PA) BX-60 microscope equipped with a Qimaging (Surrey, BC Canada) Retiga EXi monochrome CCD camera, a Lumencor AURA LED light source, Semrock (Rochester, NY) GFP-3035B and mCherry-A-000 filter sets, and a Tofra (Palo Alto, CA) focus drive, run using micromanager 2.0ß software (Edelstein et al., 2014) using a 40X air lens at 20% LED power with 50 ms exposures. PLM soma and ALM soma signals were quantified using the FIJI version of ImageJ software (Schindelin et al., 2012) as described in Nonet (2020).

Plasmid constructions
Integration vectors were assembled using Golden Gate (GG) reactions as described in Nonet (2020). Other plasmids were constructed using standard cloning techniques.

NMp3401 DR274 CT linker
NMp3055 was digested with EcoRI and HindIII and the double stranded (ds) oligonucleotide NMo5948/49 was ligated into the vector.

NMp3403 DR274 CT-FP linker
NMp3055 was digested with EcoRI and HindIII and the ds oligonucleotide NMo5952/53 was ligated into the vector.